Method for soybean transformation and regeneration

ABSTRACT

A method for Agrobacterium-mediated transformation and regeneration of soybean is disclosed. The method utilizes a cotyledon explant which is prepared by first removing the hypocotyl and then tearing the two cotyledons apart at the cotyledonary node. The explant may be inoculated with either a smear of the disarmed Agrobacterium vector or of liquid culture of the bacterium.

This application is a divisional of U.S. Ser. No. 08/388,642 filed Feb.14, 1995, now U.S. Pat. No. 5,569,834, which is a Divisional of U.S.Ser. No. 08/156,611 filed Nov. 23, 1993, now U.S. Pat. No. 5,416,011,which is a Continuation of U.S. Ser. No. 08/018,347 filed Feb. 16, 1993,now abandoned, which is a Continuation of U.S. Ser. No. 07/223,147 filedJul. 22, 1988 now abandoned.

The present invention relates to plant cell transformation andregeneration into a differentiated transformed plant. More particularly,the invention relates to a method for transforming soybean (Glycine max)using Agrobacterium-mediated transformation of a plant tissue explantand subsequent regeneration of the transformed cells into a whole plant.

BACKGROUND OF THE INVENTION

Soybean is grown on approximately 34 million hectares in the UnitedStates and Brazil. Unfortunately, only a few plant introductions havegiven rise to the major cultivars grown in the United States and, as aconsequence, this narrow germplasm base has limited soybean breeding.Hence, modification of soybean using genetic engineering techniqueswould facilitate the development of new varieties with traits such asherbicide resistance, disease resistance such as virus resistance andseed quality improvement in a manner unattainable by traditionalbreeding methods or tissue-culture induced variation. Genes have beentransferred to soybean protoplasts by electroporation of free DNA(Christou et al., 1987 and Lin et al., 1987). However, regenerationtechnology for soybean has not progressed to a state where regeneratedplants can be produced from protoplasts. Soybean shoot organogenesisoccurs from tissues such as cotyledonary nodes (Cheng et al., 1980;Wright et al., 1986; and Barwale et al., 1986) as well as primary leavesof seedlings (Wright et al., 1987). Somatic embryogenesis has beendemonstrated from immature embryos and cotyledons of developing seeds(Ranch et al., 1985; Lazzeri et al., 1985; Ghazi et al., 1986; Barwaleet al., 1986; and Hammat et al., 1987).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a map of plasmid pMON9749.

FIG. 2 shows a map of plasmid pMON894.

FIGS. 3a-3b show a diagrammatic representation of the cotyledon explantused in the present method.

SUMMARY OF THE INVENTION

The present invention provides a method for transformation of soybeanexplants and regeneration of the transformed explant into adifferentiated transformed plant.

In one aspect, the present invention embraces the discovery of a novelsoybean explant which enables Agrobacterium tumefaciens mediatedtransformation of cells which are competent to regenerate into wholetransformed plants. In another aspect the present invention embraces amethod for transforming soybeans which comprises:

(a) preparing a cotyledon explant from a soybean seedling by:

(i) removing the hypocotyl region by cutting just below the cotyledonarynode,

(ii) separating the two cotyledons at the cotyledonary node by tearingthe cotyledons apart, and

(iii) removing the epicotyl from the cotyledon to which it is attached,

(b) inserting a chimeric gene into the explant of part (a) byinoculation and co-cultivation of the explant with a disarmedAgrobacterium tumefaciens vector containing said chimeric gene;

(c) selecting transformed explant tissue, and

(d) regenerating a differentiated transformed plant from the transformedexplant tissue of part (c).

In yet another aspect, the present invention embraces a method furthercomprising wounding the cotyledon explant prior to inoculation by makingat least one cut in the petiole region (which includes the axillary bud)of the explant.

Transgenic soybean plants have been obtained within 3-4 months by aprocedure which uses Agrobacterium-mediated DNA transfer. The transgenicnature of these R_(o) plants was confirmed by expression of introducedtraits (glucuronidase, GUS; neomycin phosphotransferase, NPTII; and/or aglyphosate tolerant EPSP synthase) in leaves and in progeny. Southernhybridization analysis of the progeny of one pMON9749 (GUS) plant showedsegregation for the expected DNA fragments. The development of thepreferred embodiment of the present soybean transformation protocolrelied upon the variation of several parameters such as soybeancultivar, Agrobacterium strain, explant source, and antibioticselection. Optimization of these parameters was facilitated by the useof the GUS histochemical marker, since the effect of differenttreatments could be assessed in tissues long before plantlets could beanalyzed.

The cultivars which were used in the exemplary embodiments of thesoybean transformation protocol provided hereinafter were thosedetermined to be most susceptible to Agrobacterium transformation. Thisis important since soybean is a relatively poor host for Agrobacterium.It had previously been reported that only 2.5% of nearly 1000 soybeancultivars were susceptible to tumor induction by Agrobacterium (Wang etal., 1983). However, the transformation of soybean cells does not alwaysresult in the formation of a tumor (Faccioti et al., 1985). An in vitroscreen to identify susceptible soybean genotypes was developed based onthe ability of cultivars to produce kanamycin resistant callus afterAgrobacterium transformation. The A. tumefaciens strain A208 carryingthe disarmed nopaline plasmid pTiT37-SE (EPO Publ. No. 218,571) was thevector chosen for this screen since A208 is highly virulent on soybean(Byrne et al., 1987). The cultivars Delmar, Maple Presto and Pekingproduced significantly more kanamycin resistant callus than did othercultivars after transformation with A. tumefaciens strainpTiT37-SE::pMON273. The cultivar Peking had been previously identifiedas a genotype which was susceptible to Agrobacterium based on tumorformation (Owens et al., 1985; Byrne et al., 1987), and this cultivar'sin vitro response to Agrobacterium correlated with its in vivo response.

The cotyledon regeneration system proved to be an excellent vehicle forthe production of transgenic soybean plants. Shoot formation was rapidand prolific, and a large proportion of these shoots developed intofertile plants. In addition, this explant allowed Agrobacteriumtransformation to be targeted to regeneration competent tissue. Otherregeneration systems for soybean such as cotyledonary node (Cheng etal., 1980; Wright et al., 1986; Barwale et al., 1986), primary leaf(Wright et al., 1987) and immature embryo (Ranch et al., 1985; Lazzeriet al., 1985; Ghazietal, 1986; Barwale et al., 1986; and Hammat et al.,1987) have not yet yielded transgenic plants via Agrobacterium-mediatedtransformation. In these systems, Agrobacterium transformation may betargeted to cells which do not readily regenerate. Transformation ofprotoplasts have been transformed by both Agrobacterium (Baldes et al.,1987) and electroporation of free DNA (Christou et al., 1987 and Lin etal., 1987). Unfortunately, plant regeneration from soybean protoplastsis not yet possible.

Selection of transformed tissue is most often accomplished in planttransformation by inserting an antibiotic resistant gene into thetransformed tissue. Numerous antibiotics have been demonstrated to beeffective in plant transformation including, but not limited to,gentamicin, kanamycin, hygromycin as well as methotrexate anon-antibiotic selection agent. For purposes of the present inventionkanamycin is preferred. Kanamycin selection enriched for the transformedcell population and facilitated the production of transgenic soybeanshoots. Visualization of GUS in transformed cells clearly demonstratedthat soybean cells transformed with pMON9749 grew better with kanamycinthan without. This selection aided in the production of transgenicshoots by allowing transformed cells to grow into tissues capable ofinitiating multicellular shoot primordia. Kanamycin resistance hasproven to be a nearly universal selectable marker for transformed plantcells since it has been effective in the transformation of plant speciesas diverse as oil seed rape (Fry et al., 1987), lettuce (Michelmore etal., 1987) and corn (Rhodes et al., 1988).

Construction of Agrobacterium tumefaciens mediated transformationvectors is well known in the art, see for example Rogers et al., 1986;Rogers et al., 1987a; Rogers et al., 1987b; and Deblaere et al., 1987.The Agrobacterium-mediated transformation vectors can be used to inserta selected chimeric plant gene an explant susceptible to infection bythe Agrobacterium host.

Briefly, the gene comprises a promoter, structural coding sequence and a3' polyadenylation signal. Promoters which are known or found to causetranscription of the EPSPS gene in plant cells can be used in thepresent invention. Such promoters may be obtained from plants or virusesand include, but are not necessarily limited to, the 35S and 19Spromoters of cauliflower mosaic virus and promoters isolated from plantgenes such as EPSPS, ssRUBISCO genes and promoters obtained from T-DNAgenes of Agrobacterium tumefaciens such as nopaline and mannopinesynthases. The particular promoter selected should be capable of causingsufficient expression to result in the desired phenotypic trait. The RNAproduced by the gene also contains a 5' non-translated leader sequence.This sequence may be derived from any gene and may be specificallymodified so as to increase translation of the mRNA. The 5'non-translated regions may be derived from viral RNAs, other suitableeukaryotic genes or a synthetic gene sequence. It may be part of the 5'end of the non-translated region of the structural coding sequence forthe encoded polypeptide or derived from an unrelated promoter or codingsequence as discussed above. The 3' non-translated region contains apolyadenylation signal which functions in plants to cause the additionof polyadenylate nucleotides to the 3' end of the mRNA. In cases wherethe structural coding sequence is derived from a plant source one canuse the 3' non-translated region naturally associated with theparticular plant gene. Examples of other suitable 3' regions are the 3'transcribed, non-translated regions containing the polyadenylationsignal of the nopaline synthase (NOS) gene of the Agrobacteriumtumor-inducing (Ti) plasmid or the conglycinin (7S) storage proteingene.

The genetic modification of soybean through biotechnology has greatagricultural value. Soybean is a major food and feed source which isgrown on more acres worldwide than any other dicotyledonous crop. Thelimited genetic base in domestic soybean varieties has limited the powerof traditional breeding methods to develop varieties with improved orvalue-added traits. The development of herbicide resistant soybeancultivars would provide simpler and more effective weed control insoybean fields. The glyphosate resistant soybean plant produced usingthis soybean transformation protocol is an example of the speed in whichnew agronomic traits can now be introduced into soybean cultivars.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Numerous soybean cultivars, chosen for their relative diversity, werescreened for their in vitro response to A. tumefaciens mediatedtransformation. All cultivars grew callus on MS NAA/K medium. Thismedium contains MS salts and organics (Murashige and Skoog, 1962) with2.15 mg/l kinetin and 4.68 mg/l napthalene acetic acid (NAA). Therefore,this medium was chosen for use in an in vitro screen for A. tumefacienssusceptibility based on the production of antibiotic resistant callus.The susceptibility was determined by scoring the number of soybeanhypocotyls which were capable of producing callus on MS NAA/K mediumcontaining 100 mg/l kanamycin after inoculation and co-culture with A.tumefaciens pTiT37-SE::pMON273 (Rogers et al., 1987 and Sanders et al.,1987). The intermediate plasmid pMON273, which contains a chimericneomycin phosphotransferase II (NPTII) gene with the cauliflower mosaicvirus (CaMV) 35S promoter and the nopaline synthase (NOS) 3'polyadenylation signal, confers resistance to the antibiotic kanamycin.Hypocotyl explants transformed with A. tumefaciens pTiT375-SE::pMON120(Fraley et al., 1985), a construct which does not confer kanamycinresistance, never callused on MS NAA/K medium containing kanamycin.

Soybean seeds of about one hundred different soybean cultivars and plantintroductions were aseptically germinated for 4 days on 0.8% Difcopurified agar. Hypocotyls were cut into 5 mm segments, and inoculated bysmearing the cut ends of the hypocotyls with A. tumefacienspTiT37-SE::pMON273. The hypocotyl segments were co-cultured with theAgrobacterium vector for two days on 1/10 SH (Shenk and Hildebrandt)medium (1/10 the major and minor salts of SH, Gamborg et al., 1968)prior to being placed on MS NAA/K medium containing 500 mg/lcarbenicillin with or without 100 mg/l kanamycin. MS NAA/K medium iscomposed of MS salts and organics (Schenk et al., 1972) with 2.15 mg/lkinetin and 4.68 mg/l napthalene acetic acid (NAA). Each cultivar samplewas represented by 20-40 segments. The hypocotyl segments remained on MSNAA/K for 4 weeks prior to scoring. The number of hypocotyls whichproduced callus were counted as well as the number of independent calliper explant.

The callusing responses for seven cultivars of the over one hundredcultivars screened are shown in Table I below in order to show the rangein cultivar response. Specifically, Table I shows the callusing responseof soybean hypocotyl segments after inoculation and co-culture with A.tumefaciens pTiT37SE::pMON273 and subsequent culture on MS NAA/K mediumcontaining 100 mg/l kanamycin.

                  TABLE I                                                         ______________________________________                                                   Total       Number   Number                                        Cultivar   Number      Callusing                                                                              Calli                                         ______________________________________                                        Maple Presto                                                                             35          13       22                                            Peking     35          13       19                                            Delmar     30          10       18                                            Pl 181537  35          7        10                                            Cutler 71  35          5         5                                            Altona     28          3         5                                            Hartz 5370 18          0         0                                            ______________________________________                                    

The cultivars Maple Presto and Peking were identified as the mostresponsive cultivars and were used in subsequent transformationexperiments. Although some cultivars did not exhibit a callusingresponse in the above screen (e.g. Hartz 5370), this observation isbelieved to be the result of low frequency. There is no expectancy thatthese cultivars could not be transformed and regenerated by the methoddescribed herein albeit probably at a lower frequency than the moreresponsive cultivars.

Cotyledon explants did not demonstrate efficient selection for kanamycinresistant callus as did the smaller hypocotyl explants. To determine theefficacy of Agrobacterium transformation of cotyledon explants and todetermine if transformation could be targeted to the tissues competentto regenerate, the β-glucuronidase (GUS) gene was used as ahistochemical marker (Jefferson, 1987). The vector pMON9749 (FIG. 1) isan integrating plant transformation vector in which the GUS gene isdriven by the 35S promoter of cauliflower mosaic virus and contains theNOS 3' polyadenylation signal. pMON9749 was constructed as follows: AHindIII/EcoRI fragment of pRAJ260 (Jefferson et al., 1986) containingthe GUS coding sequence was inserted into HindIII/EcoRI digestedBluescript KS+(Stratagene, La Jolla, Calif.) resulting in pMON9948. Thegene was removed from pMON9948 on a ClaI/EcoRI fragment and was insertedinto pMON316 (Rogers et al., 1987b) that had been digested with the sameenzymes. NOS-NPTII-NOS, a marker allowing for selection of kanamycinresistant plant cells; Tn7 Spec R, bacterial spectinomicin resistancegene from transposon Tn7; RB, the right border of the pTiT37 T-DNA; 35S,the 35S promoter from cauliflower mosaic virus; 3', the poly-(A)addition region of the nopaline synthase gene.

The vector was integrated into the disarmed Ti plasmid A. tumefacienspTiT37-SE (EPO Publ. No. 218,571). Soybean cotyledon explants wereinoculated with A. tumefaciens pTiT37-SE::pMON9749 and assayed for GUSactivity after 3 weeks of culture on B₅ BA medium containing kanamycin200-300 mg/l. B₅ BA medium is composed of B₅ salts (Gamborg et al.,1968), 20 mg/l sucrose, 1.15 mg/l benzyladenine (BA), 8 g/l Difcopurified agar at pH 5.8 prior to autoclaving. Transformed cells wereidentified after the hydrolytic action of the GUS enzyme converted thesubstrate 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) into aninsoluble blue precipitate in the cell cytoplasm (Jefferson, 1987). Infree hand sections of the cotyledons, multiple GUS-positive callussectors were identified in callus associated with the excision woundsite. Some of these GUS-positive callus sectors were observed in theregeneration competent region of cotyledons adjacent to an incipientshoot primordium. The utility of the cotyledon explant in targetingAgrobacterium-mediated transformation to the regenerating cellpopulation was more clearly demonstrated when several GUS positive shootprimordia were found. Similar shoot primordia and callus on cotyledonstransformed with the plasmid pMON200 (Fraley et al., 1985) without theGUS gene did not react positively after the X-Gluc reaction. Utilizingthe GUS analysis significantly sped up the development of the cotyledontransformation protocol, inasmuch as evidence for shoot transformationcould be obtained several months before regenerated plants could beanalyzed.

Briefly, in an exemplary embodiment the method of the present inventioncomprises sterilizing the seeds of a selected soybean cultivar and thengerminating the sterilized seed to provide explant material. Theprepared cotyledonary explant is inoculated with the A. tumefacienstransformation vector carrying the desired plant gene and a suitableselectable marker such as neomycin phosphotransferase II. Thetransformed cells are then selected under the appropriate antibioticpressure. The surviving explants are then moved to shoot inductionmedium. Shoots are then transferred to root induction medium to producea regenerated plant.

Seed Sterilization

The seeds are placed in a suitable sized flask (500 ml). The seeds arewashed in a dilute aqueous detergent solution for about five minutes.The seeds are then washed 5 or 6 times with distilled water. The seedsare then alcohol washed with 70 wt% ethanol for about 1-2 minutes withoccasionally shaking. The ethanol is decanted and about 200 ml of 50%Chlorox (5.25% sodium hypochlorite) containing about one drop Tween 20is added with occasionally agitation. After 5-15 minutes the Chloroxsolution is decanted and the seeds rinsed with sterile distilled waterabout 5 times or until there is no suds remaining. Enough steriledistilled water is added to cover the seeds along with a fungicidefollowing the manufacturer's directions. The flask is gently shaken afew times and the seeds are soaked for about one hour. The fungicidesolution is removed and the seeds stored in a dry, sterile petri dish.

Seed Germination

The sterile seeds of the selected soybean cultivar are germinated inpetri dishes (100×25 mm) containing B₅ O medium. B₅ O medium is the sameas B₅ BA medium but without the benzyladenine. The seeds are simply laidon top of the medium. The plates are wrapped with parafilm or placed inplastic bags. The seeds are maintained under an environment of 16 hourslight/8 hours dark, 27° C. for 4-5 days.

A. tumefaciens Preparation

The A. tumefaciens cells containing the selected transformation vectorare grown in fresh LBSCCK medium about 2-5 days before use. The cellsare grown at 25° C. LBSCCK consists of Luria Broth medium (either solidor liquid) with 50 mg/l spectinomycin, 50 mg/l streptinomycin, 25 mg/lchloramphenical and 50 mg/l kanamycin. Luria Broth liquid mediumconsists of tryptone, 10 grams/l; yeast extract, 5 grams/l; sodiumchloride, 10 grams/l at pH 7.0. Luria Broth solid consists of LuriaBroth liquid with 15 grams/l Bacto Agar.

In the case of liquid culture a loopful of bacteria is added to 2 mls ofLBSCCK medium two days prior to use. The next 2 ml of fresh medium isinoculated with 100-200 μl of the previous culture. For explantinoculation the next day the cells are spun down, washed with SHO mediumand resuspended in an equal volume of SHO medium. For liquid inoculationabout 10-16 ml of culture is preferably used per 10 cotyledon explants.In the case of smear inoculation preferably one plate of A. tumefacienscells are used per 40 cotyledonary explant.

Explant Preparation/Inoculation

Referring to FIG. 3, the cotyledon explant is prepared in the followingmanner. The seedlings are cut off about 3 mm below the cotyledons andput into a sterile empty plate. A small cut is made between the twocotyledons using straight forceps and a scalpel blade, and then the twocotyledons are ripped apart. The epicotyl is removed with forceps. Thecotyledons are wounded by making 5-7 slight horizontal cuts in thepetiole region (including the axillary bud area), one should be carefulnot to cut through the entire cotyledon. After preparing the explant asdescribed above, it is put into the 1/10 SHO plate (SH medium withouthormones, containing 200 μM Acetosyringone), preferably with thecotyledon positioned adaxial side up.

The end of the straight edge of the bacterial loop is rubbed across thesurface of the bacterial plate a few times. The loop is carefullysmeared in the axillary bud area of the wounded cotyledon. Thisprocedure is repeated for each cotyledon, keeping the cover closed onthe 1/10 SHO plate as much as possible. In the case of liquidinoculation, the explants are soaked in the bacteria solution for aboutone-half hour, blotted onto sterile filter paper and transferred to 1/10SHO plates. It has been found that in many cases higher frequencies areobtained if the part of the explant away from the petiole is dug intothe medium at a slant so the petiole is not in contact with the surfaceof the medium. Plates are wrapped with parafilm 12 cm by 5 cm folded inthirds, and put in same percival as germinated seeds in. Transfer in 3-4days.

Kanamycin Selection

Cotyledons are moved to a dish containing B₅ -2.5-10 μM BA with 500 mg/lcarbenicillin and 100 mg/l cefotaximine and 250 mg/l kanamycin.Cotyledons explant are put adaxial side in contact with the medium,without digging them into the medium. The inoculated cotyledons aremaintained at 25° C. under a photoperiod of 16:8 (cool white fluorescentlight at 40 uEn/s).

After four weeks all cotyledons are moved to B₅ O with 500 mg/lcarbenicillen and 100 mg/l cefotaxime and 250 mg/l kanamycin. Thepetiole is preferably dug into the medium so the regenerating shoots arein contact with the medium. The cotyledon may be sideways or sticking upin the air. The plates are wrapped in parafilm. Explants producingshoots are subcultured every 4 weeks into fresh B₅ O medium. Cotyledontissue that becomes necrotic is removed upon subculturing.

Root Induction/Plantlet Formation

Elongating shoots (1-2 inches) were removed and placed on 1/2 B₅ Omedium (half the major and minor salts of B₅ O medium) in capped glassvials or in sterile 50 ml disposable plastic centrifuge tubes. Plantlets(rooted shoots) were moved to vermiculite in 2 inch pots after severalnew leaves had been produced. These plantlets were placed in a plasticcontainer in which the lid was gradually opened to harden them off priorto growing in the greenhouse. Plantlets which had produced new leavesafter hardening off were transplanted into soil and grown in thegreenhouse for flowering and seed set.

EXAMPLE 1

Transgenic soybean plants which express β-glucoronidase was producedfrom explants of cultivars Peking and Maple Presto prepared in themanner described above. Cotyledon explants were prepared from seedlingsobtained from germinated sterile seeds. The explant was inoculated witha smear of A. tumefaciens 208 containing pTiT37-SE::pMON9749. PlasmidpMON9749 (FIG. 1) contains the β-glucuronidase plant gene(CaMV35S/β-glucuronidase/NOS3') as well as the selectable neomycinphosphotransferase marker gene (NOS/NPTII/NOS). Transformed explanttissue was selected on media containing 200-300 mg/l kanamycin. Shootsfrom kanamycin resistant tissue were rooted and plantlets obtained.

Table II below lists results from three experiments involvingtransformation using pMON9749. Plantlets were determined to betransgenic by three different assays. Transgenic pMON9749 plants hadleaves which were positive in the GUS histochemical reaction and theNPTII dot blot assay in addition to producing callus on MS19 mediumcontaining 100 mg/l kanamycin.

β-glucuronidase (GUS) enzyme activity was located histochemically inunfixed free hand sections as described by Jefferson, 1987. After thehistochemical reaction was complete, the sections were fixed in FAA for1 day and cleared in 70% ethanol. The presence of nopaline was assayedby paper electrophoresis, Murashige and Skoog, 1962. Neomycinphosphotransferase II (NPTII) activity was determined by the dot blotprocedure described by McDonnell et al., 1987. Kanamycin resistance wasassayed by the ability of leaf tissue to produce callus on MS19 mediumcontaining 500 mg/l carbenicillin, 100 mg/l cefotaxime and 100 mg/lkanamycin. MS19 medium is composed of MS salts and organics with 2 mg/lBA and 0.5 mg/l NAA. Whole or cut leaflets were placed on the medium,and if callusing occurred within 4 weeks, they were scored as resistant.Nontransgenic leaf tissue failed to callus on this medium.

                  TABLE II                                                        ______________________________________                                                 Number                                                                        Regenerating  Number of                                                                              Number                                        Construct                                                                              Total         Plantlets                                                                              Transgenic                                    ______________________________________                                        9749      7/43         6         1.sup.b                                       9749.sup.a                                                                             6/130        13       1                                             9749     18/88         6        2                                             ______________________________________                                         .sup.a This experiment utilized the cultivar Maple Presto                     .sup.b Progeny analysis, infra.                                          

EXAMPLE 2

Transgenic soybean plants which express a mutant petunia5-enolpyruvylshikimatephosphate synthase (EPSP synthase) tolerant towardglyphosate herbicide were produced from explants of the cultivar Pekingin the manner described above. Construction of mutant EPSP synthaseplant genes is described in commonly assigned U.S. patent applicationsSer. No. 879,814, filed Jul. 7, 1986 and Ser. No. 179,245, filed Apr.22, 1988, the disclosures of which are incorporated by reference herein.Cotyledon explants were prepared from seedlings obtained from germinatedsterile seeds. The explant was inoculated with a smear of A. tumefaciens208 containing pTiT37-SE::pMON894. Plasmid pMON894 (FIG. 2 contains themutant petunia EPSP synthase gene, CaMV35S/Mutant petuniaCTP/EPSPS/NOS3', as well as the selectable neomycin phosphotransferaseII marker gene (NOS/NPTII/NOS). Transformed explant tissue was selectedon medium containing 200-300 mg/l kanamycin. Shoots from kanamycinresistant tissue rooted and plants obtained. Table III below listsresults obtained from three experiments involving the transformation ofthe Peking cultivar using pMON894. Transgenic pMON894 plants werepositive in the NPTII dot blot assay, produced callus on MS19 mediumcontaining 100 mg/l kanamycin and produced leaves which callused on MS19medium containing 1.0 mM glyphosate.

                  TABLE III                                                       ______________________________________                                                 Number                                                                        Regenerating  Number of                                                                              Number                                        Construct                                                                              Total         Plantlets                                                                              Transgenic                                    ______________________________________                                        894       21/242       35       1                                             894       43/247       13        1.sup.b                                      894      194/650       55       2                                             ______________________________________                                         .sup.b Progeny analysis, infra.                                          

Leaves of a transgenic soybean plant containing pMON894 displayed ahigher level (4-fold) of EPSP synthase activity over that of wild typecontrol leaves (Table IV). In addition, the EPSP synthase activity ofthe transgenic plant was unaffected by 0.5 mM glyphosate. Under theseconditions, there is a complete inhibition of the endogenous EPSPsynthase activity.

                  TABLE IV                                                        ______________________________________                                        EPSP Synthase Specific Activity.sup.1                                         Plant         0 mM    0.5 mM Glyphosate                                       ______________________________________                                        Wild Type      7       0                                                      pMON894       30      30                                                      ______________________________________                                         .sup.1 Expression of a petunia mutant EPSP synthase cDNA in a transgenic      soybean plant leaf. EPSP synthase activity is expressed as the specific       activity (nmol of EPSP formed/min mg/protein). Enzyme assays were             performed in the presence of 0.5 mM glyphosate in order to completely         inhibit the endogenous EPSP synthase activity and measure the mutant EPSP     synthase activity resulting from the gene in pMON894.                    

Analysis of Transgenic Progeny

Of 14 selfed R₁ progeny from a pMON9749 transgenic soybean plant, 11co-segregated for GUS and NPT activity. This is approximately a 3:1segregation ratio indicating the presence of a single active T-DNAlocus. Southern analysis confirmed that these progeny plants containedthe inserted DNA fragment necessary to confer these genetic traits.Southern hybridization was performed on 6 of the R₁ progeny to assay forthe presence and copy number of pMON9749 T-DNA in the plants. Two of theprogeny analyzed were negative for GUS and NPTII, the other four werepositive. Genomic DNA digested with HindIII was hybridized with alabeled pMON9749 probe. The four R₁ plants that were enzyme positiveshowed strong hybridization with the probe at a level consistent withone or a few copies of the T-DNA. All of the hybridizing plants showedthe same pattern of putative T-DNA junction fragments indicating thatthere are no silent copies of the T-DNA segregating independently of theactive copy. The junction fragment pattern is consistent with a singlesite of T-DNA insertion. This positive hybridization result and thecorrelation between enzyme activity and T-DNA in the R₁ progeny areevidence that this pMON9749 transgenic soybean plant was generated bythe expected Agrobacterium-mediated events. A pMON894 plant alsoproduced progeny which co-segregated in a 3:1 ratio for kanamycinresistance and glyphosate tolerance.

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We claim:
 1. A method for transforming soybean which comprises:(a)preparing a cotyledon explant from a soybean seedling by:(i) removingthe hypocotyl region by cutting just below the cotyledonary node, (ii)separating the two cotyledons at the cotyledonary node by tearing thecotyledons apart, and (iii) removing the epicotyl from the cotyledon towhich it remains attached, (b) inserting a chimeric gene into theexplant of part (a) by inoculation and co-cultivation of the explantwith a disarmed Agrobacterium tumefaciens vector containing saidchimeric gene; (c) selecting transformed explant tissue, and (d)regenerating a differentiated transformed plant from the transformedexplant tissue of part (c).
 2. The method of claim 1 wherein thecotyledon explant is wounded prior to inoculation with the Agrobacteriumtumefaciens vector by making at least one cut in the petiole region ofthe explant.
 3. The method of claim 1 wherein transformed explant tissueis selected by resistance to kanamycin antibiotic.
 4. The method ofclaim 3 in which the concentration of kanamycin is between 200 and 300mg/l.
 5. The method of claim 1 in which the vector is Agrobacteriumtumefaciens A208 carrying plasmid pTiT37-SE.
 6. The method of claim 2 inwhich the vector is Agrobacterium tumefaciens A208 carrying plasmidpTiT37-SE.
 7. The method of claim 3 in which the vector is Agrobacteriumtumefaciens A208 carrying plasmid pTiT37-SE.
 8. The method of claim 4 inwhich the vector is Agrobacterium tumefaciens A208 carrying plasmidpTiT37-SE.
 9. The method of claim 1 in which the soybean is a cultivarselected from the group consisting of Peking and Maple Presto.
 10. Amethod for transforming soybean which comprises:a) preparing a cotyledonexplant from a soybean seedling by:i) removing the hypocotyl region bycutting just below the cotyledonary node, ii) separating the twocotyledons at the cotyledonary node by tearing the cotyledons apart, andiii) removing the epicotyl from the cotyledon to which it is attached,and iv) wounding the explant by making at least one cut in the axillarybud region of the explant, b) inserting a chimeric gene into the explantof part (a) which gene encodes for 5-enolpyruvylshikimatephosphatesynthase by inoculation and co-cultivation of the explant with adisarmed Agrobacteriun tumefaciens vector containing said chimeric gene;c) selecting transformed explant tissue by growing the explant in thepresence of glyphosate, and d) regenerating a differentiated transformedplant from the transformed explant tissue of part (c).
 11. The method ofclaim 2 in which the soybean is transformed to express a glyphosatetolerant 5-enolpyruvylshikimatephosphate synthase.
 12. The method ofclaim 11 in which the vector is Agrobacterium tumefaciens A208containing pTiT37-SE::pMON894.
 13. The method of claim 10 in which thevector is Agrobacterium tumefaciens A208 containing pTiT37-SE::pMON200.14. A soybean plant produced by the method of claim
 1. 15. A transformedsoybean plant produced according to claim 1 which contains a chimericgene encoding a glyphosate-tolerant 5-enolpyruvylshikimatephosphatesynthase.
 16. A transformed soybean plant produced by the method ofclaim
 2. 17. A transformed soybean plant produced by the method of claim10.
 18. A seed produced from the plant of claim
 14. 19. A seed producedfrom the plant of claim 15.